Methods of producing singlet oxygen using compounds having improved functionalization

ABSTRACT

Novel texaphyrin compounds having improved functionalization are described. Metal complexes of these compounds are active as photosensitizers for the generation of singlet oxygen and thus are potentially useful for treatments performed with singlet oxygen. Several of the metallotexaphyrin complexes absorb light in the physiologically important range of 690-880 nm. The complexes form long-lived triplet states and thus may act as efficient photosensitizers for generation of singlet oxygen.

This application is a continuation application of U.S. application Ser.No. 08/459,333, filed Jun. 2, 1995, now U.S. Pat. No. 5,599,929, theentire text of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of expanded porphyrins, inparticular, to texaphyrins having improved functionalization.

BACKGROUND OF THE INVENTION

Certain texaphyrin compounds are described in U.S. Pat. Nos. 4,935,498,5,162,509, 5,252,720, 5,272,142 and 5,256,399, each of which isincorporated by reference herein. "Texaphyrin" refers to a particular"expanded porphyrin" pentadentate macrocyclic ligand. The compound iscapable of existing in both its free-base form and of supporting theformation of a 1:1 complex with a variety of metal cations, such asCd²⁺, Hg²⁺, In³⁺, y³⁺, Nd³⁺, Eu³⁺, Sm³⁺, La³⁺, Lu³⁺, Gd³⁺, and othercations of the lanthanide series that are too large to be accommodatedin a stable fashion within the 20% smaller tetradentate binding core ofthe well-studied porphyrins.

Large, or "expanded" porphyrin-like systems are of interest for severalreasons: They could serve as aromatic analogues of the better studiedporphyrins or serve as biomimetic models for these or other naturallyoccurring pyrrole-containing systems. In addition, largepyrrole-containing systems offer possibilities as novel metal bindingmacrocycles. For instance, suitably designed systems could act asversatile ligands capable of binding larger metal cations and/orstabilizing higher coordination geometries than those routinelyaccommodated within the normally tetradentate ca. 2.0 Å radius porphyrincore. The resulting complexes could have important application in thearea of heavy metal chelation therapy, serve as contrast agents formagnetic resonance imaging (MRI) applications, act as vehicles forradioimmunological labeling work, or serve as new systems for extendingthe range and scope of coordination chemistry.

The desirable properties of texaphyrins are:

1) appreciable solubility, particularly in aqueous media;

2) biolocalization in desired target tissue;

3) low intrinsic toxicity;

4) the ability to attach to solid matrices;

5) the ability to be attached to biomolecules;

6) efficient chelation of divalent and trivalent metal cations;

7) absorption of light in the physiologically important region of690-880 nm;

8) high chemical stability;

9) ability to stabilize diamagnetic complexes that form long-livedtriplet states in high yield and that act as efficient photosensitizersfor the formation of singlet oxygen;

10) ability to chelate Gd(III) for magnetic resonance imaging;

11) a redox potential lower than that of oxygen for use as aradiosensitizer.

One of the disadvantages of the texaphyrin metal complexes of priorpatents is their short half-life. The Y³⁺ and In³⁺ complexes of thebasic texaphyrin have half-lives for decomplexation and/or liganddecomposition of about 3 weeks in 1:1 methanol-water mixtures. Whilesuch stability is adequate for some in vitro or in vivo applications, agreater degree of stability in aqueous solution is desirable. Forexample, a desired solution-phase shelf life of 2-3 years wouldfacilitate the formulation of texaphyrin metal complexes aspharmaceutical products. The new molecules of the present inventionaddress the problems of demetallation of the texaphyrin metal complexand the susceptibility of the imine bonds of the macrocycle tohydrolysis. The solution to these problems is expected to provide atexaphyrin which has a more desirable shelf life.

SUMMARY OF THE INVENTION

The present invention seeks to solve the above problems by providingtexaphyrin metal complexes having improved functionalization compared tothose previously described. The improved functionalization is two-fold:firstly, addition of electron-donating groups to positions 2, 7, 12, 15,18 and/or 21 of the macrocycle contributes electrons to the aromatic πsystem of the macrocycle which stabilizes the metal complex todemetallation and stabilizes the imine bonds to hydrolysis; andsecondly, the addition of electron-withdrawing groups to positions 15and/or 18 renders the macrocycle more readily reduced, i.e. the redoxpotential will be lower and the macrocycle will more readily gain anelectron to form a radical. The addition of substituents to the 12 and21 positions of the macrocycle also offers steric protection for theimine bonds against possible in vivo enzyme hydrolysis. Thus, themacrocycles of the present invention represent molecules where anattempt has been made to optimize their structure and properties interms of imine bond stabilization and low redox potential, propertiesthat are expected to be important for radiosensitization as well asother applications.

Exemplary electron-donating groups that may be employed in the practiceof the invention include, among others, amino, alkylamino, hydroxyl,acylamino, alkoxy, acyloxy, alkyl, aryl, and alkenyl.Electron-withdrawing groups include halide other than iodide, haloalkylother than iodoalkyl, formyl, acyl, carboxylic acid, ester, acylchloride, sulfonic acid, and nitro, among others. Other potentialelectron-donating or withdrawing groups will be apparent to one of skillin the art in light of the present disclosure.

In certain embodiments, the present invention provides a texaphyrinhaving the structure: ##STR1## M is H, a divalent metal cation, or atrivalent metal cation. R₁ -R₄, R₇ and R₈ are independently hydrogen,halide, hydroxyl, alkyl, aryl, haloalkyl, nitro, formyl, acyl,hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, saccharide, carboxy,carboxyalkyl, carboxyamidealkyl, a site-directing molecule, a catalyticgroup, or a couple to a site-directing molecule or to a catalytic group.

R₆ and R₉ are independently selected from the groups of R₁ -R₄, R₇ andR₈, with the proviso that the halide is other than iodide and thehaloalkyl is other than iodoalkyl.

R₅ and R₁₀ -R₁₂ are independently hydrogen, alkyl, aryl, hydroxyalkyl,oxyalkyl, oxyhydroxyalkyl, carboxyalkyl, carboxyamidealkyl or a coupleto a saccharide, to a site-directing molecule or to a catalytic group.

For this invention, at least one of R₅, R₆, R₉, R₁₀, R₁₁ and R₁₂ isother than hydrogen.

The charge, Z is an integer value less than or equal to 5. Here, aswould be apparent to one skilled in the art, the charge Z would beadjusted so as to account for the choice of metal, M, the pH underconsideration, and the substituents R₁ -R₁₂. For instance, if R₁=carboxyl and R₂ -R₁₂ =alkyl and the metal M=Gd⁺³, and the solution ispH=7 (so that R₁ =CO₂ -), the charge Z would be zero. The charge wouldbe negative when substituents have a sufficient number of negativecharges, for example, when a substituent is an oligonucleotide. Thecharge would be +5, for example, when the M is Gd⁺³ and the net chargeof a substituent(s) is three positive charges.

An aspect of the present invention is an embodiment where a substituentmay be an electron-donating group. In this case, R₁ -R₄ and R₆ -R₉ areindependently hydrogen, hydroxyl, alkyl, aryl, hydroxyalkyl, oxyalkyl,oxyhydroxyalkyl, saccharide, carboxyalkyl, carboxyamidealkyl, asite-directing molecule, a catalytic group, or a couple to asite-directing molecule or to a catalytic group. R₅ and R₁₀ -R₁₂ areindependently hydrogen, alkyl, aryl, hydroxyalkyl, oxyalkyl,oxyhydroxyalkyl, carboxyalkyl, carboxyamidealkyl or a couple to asaccharide, to a site-directing molecule or to a catalytic group. Atleast one of R₅, R₆, R₉, R₁₀, R₁₁ and R₁₂ is other than hydrogen and Zis an integer less than or equal to 5.

In another embodiment of the present invention, R₆ or R₉ may have anelectron-withdrawing group. In that case, R₁ -R₄, R₇ and R₈ areindependently hydrogen, halide, hydroxyl, alkyl, aryl, haloalkyl, nitro,formyl, acyl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, saccharide,carboxy, carboxyalkyl, carboxyamidealkyl, a site-directing molecule, acatalytic group, or a couple to a site-directing molecule or to acatalytic group. R₅ and R₁₀ -R₁₂ are independently hydrogen, alkyl,aryl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, carboxyalkyl,carboxyamidealkyl or a couple to a saccharide, to a site-directingmolecule or to a catalytic group. R₆ and R₉ are independently hydrogen,halide other than iodide, formyl, acyl, carboxy, or nitro, where atleast one of R₆ and R₉ is other than hydrogen and Z is an integer lessthan or equal to 5.

A couple may be an amide, disulfide, thioether, or ether covalent bond.A site-directing molecule may have binding specificity for localizationto a treatment site.

It is contemplated that the texaphyrins of the present invention areuseful in a variety of applications including use as a photodynamictherapy agent, as a magnetic resonance imaging agent, as a radiationsensitizer, for RNA hydrolysis, and for DNA photocleavage. The use of atexaphyrin diamagnetic-metal complex having a substituent at the 2, 7,12, 15, 18 and/or 21 position and an absorption range from about 730 toabout 770 nanometers includes the following methods which take advantageof the ability of these compounds to produce singlet oxygen: i) a methodof deactivating a retrovirus or enveloped virus in an aqueous fluid, themethod comprising the steps of adding said texaphyrin metal complex tosaid aqueous fluid and exposing the mixture to light to effect theformation of singlet oxygen; ii) a method of producing light-inducedsinglet oxygen comprising subjecting said texaphyrin metal complex tolight in the presence of oxygen; iii) a method of photosensitizationcomprising photoirradiating said texaphyrin; iv) a method of DNAlight-induced photocleavage comprising placing said texaphyrin incontact with the RNA or DNA and photoirradiating said texaphyrin; and v)a method of treating a host harboring atheroma or neoplastic tissuecomprising administering to the host an effective amount of saidtexaphyrin complex, the complex exhibiting selective biolocalization inthe atheroma or neoplastic tissue relative to surrounding tissues, andphotoirradiating the texaphyrin complex in proximity to the atheroma orneoplastic tissue.

Further aspects of the present invention include the use of a texaphyrinparamagnetic-metal complex having a substituent at the 2, 7, 12, 15, 18and/or 21 position in the following methods which take advantage of thehigh relaxivity of these compounds: i) a method of enhancement ofrelaxivity comprising the administration of said texaphyrin; ii) amethod of magnetic resonance image enhancement comprising administeringto a subject an effective amount of said texaphyrin followed by MRimaging of the subject; iii) a method of detection of atheroma orneoplastic tissue in a subject comprising administering to the subjectsaid texaphyrin in an amount effective to enhance a magnetic resonanceimage and detecting the atheroma or neoplastic tissue by MR imaging ofsaid subject; iv) a method of imaging an organ in a subject comprisingadministering to the subject said texaphyrin in an amount effective toenhance a magnetic resonance image of the organ and detecting the organby MR imaging of said subject; v) a method of imaging an atheroma in asubject comprising administering to the subject said texaphyrin in anamount effective to enhance a magnetic resonance image of the atheromaand detecting the atheroma by MR imaging of said subject; and vi) amethod of RNA hydrolysis comprising placing said texaphyrin in contactwith the RNA.

A method of treating a host harboring atheroma or neoplastic tissue isalso an aspect of the present invention, such method comprisingadministering to the host as a first agent a texaphyrin detectable-metalcomplex of the present invention, said complex exhibiting selectivebiolocalization in the atheroma or neoplastic tissue relative tosurrounding tissue; determining localization sites in the host byreference to such texaphyrin-detectable metal complex; administering tothe host as a second agent a texaphyrin diamagnetic-metal complex havinga substituent at the 2, 7, 12, 15, 18 and/or 21 position and havingessentially identical biolocalization property and exhibiting theability to generate singlet oxygen upon exposure to light; andphotoirradiating the second agent in proximity to said atheroma orneoplastic tissue.

The present invention provides a method of radiation therapy for a hostharboring atheroma or neoplastic tissue, the method comprisingadministering to the host a texaphyrin of the present invention, andadministering ionizing radiation to the host in proximity to theatheroma or neoplastic tissue. The radiation may be administered eitherbefore or after administration of the texaphyrin. The texaphyrinexhibits greater biolocalization in the atheroma or neoplastic tissuerelative to surrounding tissues and has radiosensitization properties.An additional step may be included, the step being the determination oflocalization sites of the atheroma or neoplastic tissue in the host bymonitoring texaphyrin concentrations.

One skilled in the art would recognize in light of the presentdisclosure that sapphyrin-conjugated texaphyrin metal complexes may beused in methods for generating singlet oxygen. Sapphyrin compounds aredisclosed in U.S. Pat. Nos. 5,159,065 and 5,120,411 which areincorporated by reference herein.

Texaphyrin metal complexes having increased solution phase stability areexpected to be more stable in vivo. Increased stability achieved viaspecific, designed modifications of the texaphyrin skeleton could giverise to products with modified biolocalization properties. Selectivetargeting would improve the efficacy and utility of texaphyrins asdiagnostic or therapeutic agents for the range of applications discussedherein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention involves metal complexes of texaphyrins having asubstituent(s) at the 2, 7, 12, 15, 18 and/or 21 position(s) of thetexaphyrin macrocycle and the synthesis and uses thereof. Thenomenclature as used herein defines a substituent R₁₁ attached toposition 2, R₁₂ attached to position 7, R₅ attached to position 12, R₆attached to position 15, R₉ attached to position 18 and R₁₀ attached toposition 21 of the macrocycle. The following structure shows acorrelation of the IUPAC nomenclature for the positions of the atomsaround the periphery of the macrocycle with the positions of the Rgroups of the present invention. ##STR2##

Substituents at the R₆ and R₉ positions on the B (benzene ring) portionof the macrocycle are incorporated into the macrocycle by theirattachment to ortho-phenylenediamine in the 3 and 6 positions of themolecule. Substituents at the R₅ and R₁₀ positions on the T (tripyrrane)portion of the macrocycle are incorporated by appropriatefunctionalization of carboxyl groups in the 5 positions of thetripyrrane at a synthetic step prior to condensation with a substitutedortho-phenylenediamine.

In a method for synthesizing a texaphyrin metal complex having asubstituent at the 2, 7, 12, 15, 18 or 21 position, the method comprisesthe steps of: i) mixing, in an organic solvent, a nonaromatic texaphyrinhaving a substituent at the 2, 7, 12, 15, 18 and/or 21 position, atrivalent metal salt, a Br.o slashed.nsted base and an oxidant; and ii)allowing the mixture to react to form an aromatic texaphyrin metalcomplex having a substituent at the 2, 7, 12, 15, 18, and/or 21position. A preferred means is to stir at ambient temperature or heatthe mixture at reflux for at least two hours.

The corresponding nonaromatic texaphyrin having a substituent at the 2,7, 12, 15, 18, and/or 21 position is conveniently produced bycondensation of a tripyrrane aldehyde or ketone having structure A and asubstituted ortho-phenylenediamine having structure B: ##STR3##

In this embodiment, R₁ -R₄, R₇ and R₈ are independently hydrogen,halide, hydroxyl, alkyl, aryl, haloalkyl, nitro, formyl, acyl,hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, saccharide, carboxy,carboxyalkyl, carboxyamidealkyl, a site-directing molecule, or a coupleto a site-directing molecule.

R₆ and R₉ are independently selected from the groups of R₁ -R₄, R₇ andR₈, with the proviso that the halide is other than iodide and thehaloalkyl is other than iodoalkyl.

R₅, R₁₀, R₁₁ and R₁₂ are independently hydrogen, alkyl, aryl,hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, carboxyalkyl, carboxyamidealkylor a couple to a saccharide or to a site-directing molecule. At leastone of R₅, R₆, R₉, R₁₀, R₁₁ and R₁₂ is other than hydrogen.

In a preferred method of synthesis, the Br.o slashed.onsted base istriethylamine or N,N,N',N'-tetramethyl-1,8-diaminonaphthalene ("protonsponge"), and the oxidant is air saturating the organic solvent, oxygen,platinum oxide, o-chloranil or2,3-dichloro-5,6-dicyano-1,4-benzoquinone. The stirring or heating atreflux step may comprise stirring or heating at reflux the mixture forat least 24 hours. The organic solvent may comprise methanol, ormethanol and chloroform, or methanol and benzene, or methanol anddimethylformamide.

In the texaphyrins of the present invention, M is hydrogen, a divalentmetal cation, or a trivalent metal cation. The divalent metal cation maybe selected from, but is not limited to, the group consisting of Ca(II),Mn(II), Co(II), Ni(II), Zn(II), Cd(II), Hg(II), Fe(II), Sm(II) and UO₂(II). The trivalent metal cation may be selected from, but is notlimited to, the group consisting of Mn(III), Co(III), Ni(III), Fe(III),Ho(III), Ce(III), Y(III), In(III), Pr(III), Nd(III), Sm(III), Eu(III),Gd(III), Tb(III), Dy(III), Er(III), Tm(III), Yb(III), Lu(III), La(III),and U(III).

The alkyl, aryl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, saccharide,carboxyalkyl, carboxyamidealkyl, site-directing molecule, or moleculecouple is covalently bonded to the texaphyrin via a carbon-carbon, acarbon-nitrogen or a carbon-oxygen bond.

The aryl may be a phenyl group, unsubstituted or substituted with anitro, carboxy, sulfonic acid, hydroxy, oxyalkyl or halide other thaniodide substituent. In this case, the substituent on the phenyl groupmay be added in a synthetic step after the condensation step which formsthe macrocycle.

Representative examples of alkanes useful as alkyl group substituents ofthe present invention include methane, ethane, straight-chain, branchedor cyclic isomers of propane, butane, pentane, hexane, heptane, octane,nonane and decane, with methane, ethane and propane being preferred.Representative examples of alkenes useful as alkenyl group substituentsinclude ethene, straight-chain, branched or cyclic isomers of propene,butene, pentene, hexene, heptene, octene, nonene and decene, with etheneand propene being preferred. Representative examples of alkynes usefulas alkynyl group substituents include ethyne, straight-chain, branchedor cyclic isomers of propyne, butyne, pentyne, hexyne, heptyne, octyne,nonyne and decyne, with ethyne and propyne being preferred.Representative examples of substituted alkyls include alkyls substitutedby two or more functional groups as described herein.

Among the halide substituents, chloride, bromide, fluoride and iodideare contemplated in the practice of this invention with the exceptionthat R₆ and R₉ are not iodide. R₆ and R₉ may have chloride, bromide orfluoride substituents. Representative examples of haloalkyls used inthis invention include halides of methane, ethane, propane, butane,pentane, hexane, heptane, octane, nonane and decane, with halides,preferably chlorides or bromides, of methane, ethane and propane beingpreferred.

Representative examples of hydroxyalkyls include alcohols of methane,ethane, straight-chain, branched or cyclic isomers of propane, butane,pentane, hexane, heptane, octane, nonane and decane, with alcohols ofmethane, ethane or propane being preferred. "Hydroxyalkyl" is meant toinclude glycols and polyglycols having hydroxyl groups; diols of ethane,straight-chain, branched or cyclic isomers of propane, butane, pentane,hexane, heptane, octane, nonane and decane, with diols of ethane orpropane being preferred; polyethylene glycol, polypropylene glycol andpolybutylene glycol as well as polyalkylene glycols containingcombinations of ethylene, propylene and butylene.

Representative examples of oxyalkyls include the alkyl groups as hereindescribed having ether linkages. The number of repeating oxyalkylswithin a substituent may be up to 100, preferably is from 1-10, and morepreferably, is 2-3. A preferred oxyalkyl is O(CH₂ CH₂ O)_(x) CH₃ wherex=1-100, preferably 1-10, and more preferably, 2-3.

Representative examples of thioalkyls include thiols of ethane, thiolsof straight-chain, branched or cyclic isomers of propane, butane,pentane, hexane, heptane, octane, nonane and decane, with thiols ofethane (ethanethiol, C₂ H₅ SH) or propane (propanethiol, C₃ H₇ SH) beingpreferred. Sulfate-substituted alkyls include alkyls as described abovesubstituted by one or more sulfate groups, a representative example ofwhich is diethyl sulfate ((C₂ H₅)₂ SO₄).

Representative examples of phosphates include phosphate or polyphosphategroups. Representative examples of phosphate-substituted alkyls includealkyls as described above substituted by one or more phosphate orpolyphosphate groups. Representative examples of phosphonate-substitutedalkyls include alkyls as described above substituted by one or morephosphonate groups.

Representative examples of carboxy groups include carboxylic acids ofthe alkyls described above as well as aryl carboxylic acids such asbenzoic acid. Representative examples of carboxyamides include primarycarboxyamides (CONH₂), secondary (CONHR') and tertiary (CONR'R")carboxyamides where each of R' and R" is a functional group as describedherein.

Representative examples of useful amines include a primary, secondary ortertiary amine of an alkyl as described hereinabove.

Hydroxyalkyl means alkyl groups having hydroxyl groups attached.Oxyalkyl means alkyl groups attached to an oxygen. Oxyhydroxyalkyl meansalkyl groups having ether or ester linkages, hydroxyl groups,substituted hydroxyl groups, carboxyl groups, substituted carboxylgroups or the like.

Saccharide includes oxidized, reduced or substituted saccharide; hexosessuch as D-glucose, D-mannose or D-galactose; pentoses such as D-ribuloseor D-fructose; disaccharides such as sucrose, lactose, or maltose;derivatives such as acetals, amines, and phosphorylated sugars;oligosaccharides, as well as open chain forms of various sugars, and thelike. Examples of amine-derivatized sugars are galactosamine,glucosamine, sialic acid and D-glucamine derivatives such as1-amino-1-deoxysorbitol.

Carboxyamidealkyl means alkyl groups containing any number of functionalgroups, one of which is a secondary or tertiary amide. Carboxyalkylmeans alkyl groups containing any number of functional groups, one ofwhich is a carboxyl group.

For the above-described texaphyrins, oxyhydroxyalkyl may be alkyl havingindependently hydroxy substituents and ether branches or may beC.sub.(n-x) H.sub.((2n+1)-2x) O_(x) O_(y) or OC.sub.(n-x)H.sub.((2n+1)-2x) O_(x) O_(y) where n is a positive integer from 1 to10, x is zero or a positive integer less than or equal to n, and y iszero or a positive integer less than or equal to ((2n+l)-2x).

The oxyhydroxyalkyl or saccharide may be C_(n) H.sub.((2n+1)-q) O_(y)R^(a) _(q), OC_(n) H.sub.((2n+1)-q) O_(y) R^(a) _(q) or (CH₂)_(n) CO₂R^(a) where n is a positive integer from 1 to 10, y is zero or apositive integer less than ((2n+1)-q), q is zero or a positive integerless than or equal to 2n+1, R^(a) is independently H, alkyl,hydroxyalkyl, saccharide, C.sub.(m-w) H.sub.((2m+1)-2w) O_(w) O_(z), O₂CC.sub.(m-w) H.sub.((2m+1)-2w) O_(w) O_(z) or N(R)OCC.sub.(m-w)H.sub.((2m+1)-2w) O_(w) O_(z), where m is a positive integer from 1 to10, w is zero or a positive integer less than or equal to m, z is zeroor a positive integer less than or equal to ((2m+1)-2w), R is H, alkyl,hydroxyalkyl, or C_(m) H.sub.((2m+1)-r) O_(z) R^(b) _(r) where m is apositive integer from 1 to 10, z is zero or a positive integer less than((2m+1)-r), r is zero or a positive integer less than or equal to 2m+1,and R^(b) is independently H, alkyl, hydroxyalkyl, or saccharide.

Carboxyamidealkyl may be alkyl having secondary or tertiary amidelinkages or (CH₂)_(n) CONHR^(a), O(CH₂)_(n) CONHR^(a),(CH₂)NCON(R^(a))₂, or O(CH₂)_(n) CON(R^(a))₂ where n is a positiveinteger from 1 to 10, R^(a) is independently H, alkyl, hydroxyalkyl,saccharide, C.sub.(m-w) H.sub.((2m+1)-2w) O_(w) O_(z), O₂ CC.sub.(m-w)H.sub.((2m+1)-2w) O_(w) O_(z) or N(R)OCC.sub.(m-w) H.sub.((2m+1)-2w)O_(w) O_(z), where m is a positive integer from 1 to 10, w is zero or apositive integer less than or equal to m, z is zero or a positiveinteger less than or equal to ((2m+1)-2w), R is H, alkyl, hydroxyalkyl,or C_(m) H.sub.((2m+1)-r) O_(zR) ^(b) _(r) where m is a positive integerfrom 1 to 10, z is zero or a positive integer less than ((2m+1)-r), r iszero or a positive integer less than or equal to 2m+1, and R^(b) isindependently H, alkyl, hydroxyalkyl, or saccharide.

The carboxyalkyl may be alkyl having a carboxyl substituted ether, anamide substituted ether or a tertiary amide removed from an ether orC_(n) H.sub.((2n+1)-q) O_(y) R^(c) _(q) or OC_(n) H.sub.((2n+1)-q) O_(y)R^(c) _(q) where n is a positive integer from 1 to 10; y is zero or apositive integer less than ((2n+1)-q), q is zero or a positive integerless than or equal to 2n+1, Rc is (CH₂)_(n) Co₂ R^(d), (CH₂)_(n)CONHR^(d) or (CH₂)_(n) CON(R^(d))₂ where n is a positive integer from 1to 10; R^(d) is independently H, alkyl, hydroxyalkyl, saccharide,C.sub.(m-w) H.sub.((2m+1)-2w) O_(w) O_(z), O₂ CC.sub.(m-w)H.sub.((2m+1)-2w) O_(w) O_(z) or N(R)OCC.sub.(M-w) H.sub.((2m+1)-2w)O_(w) O_(z), where m is a positive integer from 1 to 10, w is zero or apositive integer less than or equal to m, z is zero or a positiveinteger less than or equal to ((2m+1)-2w), R is H, alkyl, hydroxyalkyl,or C_(m) H.sub.((2m+1)-r) O_(z) R^(b) _(r) where m is a positive integerfrom 1 to 10, z is zero or a positive integer less than ((2m+1)-r), r iszero or a positive integer less than or equal to 2m+1, and R^(b) isindependently H, alkyl, hydroxyalkyl, or saccharide.

A couple may be described as a linker, i.e., a reactive group forattaching another molecule at a distance from the texaphyrin macrocycle.An exemplary linker or couple is an amide, disulfide, thioether or ethercovalent bond as described in the examples for attachment ofoligonucleotides and antibodies. Certain reactions utilizing thetexaphyrin complexes of the present invention, such as hydrolyticcleavage of phosphate ester bonds for example, by may be enhanced byadditional catalytic groups appended to the texaphyrin metal complex orto a texaphyrin complex-site directing molecule conjugate. The term"catalytic group" means a chemical functional group that assistscatalysis by acting as a general acid, Br.o slashed.nsted acid, generalbase, Br.o slashed.nsted base, nucleophile, or any other means by whichthe activation barrier to reaction is lowered or the ground state energyof the substrate is increased. Exemplary catalytic groups contemplatedinclude, but are not limited to, imidazole; guanidine; substitutedsaccharides such as D-glucosamine, D-mannosamine, D-galactosamine,D-glucamine, and the like; amino acids such as L-histidine andL-arginine; derivatives of amino acids such as histamine; polymers ofamino acids such as poly-L-lysine, (LysAla)n or (LysLeuAla)n where n isfrom 1-30 or preferably 1-10 or more preferably 2-7, and the like;derivatives thereof; and texaphyrin metal complexes. The term "appendedto the texaphyrin-site directing molecule conjugate means that thecatalytic groups are attached either directly to the texaphyrin metalcomplex or to the texaphyrin complex via a linker or couple of variablelength, or are attached to the ligand portion of a texaphyrincomplex-ligand conjugate either with or without a linker or couple ofvariable length.

In one embodiment of the present invention, the texaphyrin is coupled tosite-directing molecules to form conjugates for targeted in vivodelivery. "Specificity for targeted sites" means that upon contactingthe texaphyrin conjugate with the targeted site, for example underphysiological conditions of ionic strength, temperature, pH and thelike, specific binding will occur. The interaction may occur due tospecific electrostatic, hydrophobic, entropic or other interaction ofcertain residues of the conjugate with specific residues of the targetto form a stable complex under conditions effective to promote theinteraction. Exemplary site-directing molecules contemplated in thepresent invention include, but are not limited to: oligonucleotides,including oligodeoxyribonucleotides and oligoribonucleotide analogs;polyamides including peptides having affinity for a biological receptorand proteins such as antibodies, low density lipoproteins, the APOprotein of lipoprotein; steroids and steroid derivatives; hormones suchas estradiol, or histamine; hormone mimics such as morphine; and furthermacrocycles such as sapphyrins and rubyrins.

Representative examples of useful oligonucleotides include nucleotides,oligonucleotides and polynucleotides primarily composed of adenine,cytosine, guanine, thymine or uracil bases. An oligonucleotide may bederivatized at the base, the sugar, the ends of the chain, or at thephosphate groups of the backbone to promote in vivo stability.Modification of the phosphate groups is preferred in one embodiment ofthe invention since phosphate linkages are sensitive to nucleaseactivity. Preferred derivatives are methylphosphonates,phosphotriesters, phosphorothioates, and phosphoramidates, and the like.Additionally, phosphate linkages may be completely substituted withnon-phosphate linkages such as amide linkages. Appendages to the ends ofthe oligonucleotide chain also provide exonuclease resistance. Sugarmodifications may include alkyl groups attached to an oxygen of a ribosemoiety in a ribonucleotide. In particular, the alkyl group generally has1 to 4 carbon atoms, and preferably is a methyl group and the methylgroup is attached to the 2' oxygen of the ribose. Other alkyl groups maybe ethyl or propyl. It is understood that the terms "nucleotide" and"oligonucleotide", as used herein, refer to both naturally-occurring andsynthetic nucleotides, poly- and oligonucleotides and to analogs andderivatives thereof.

The term "texaphyrin-oligonucleotide conjugate" means that anoligonucleotide is attached to the texaphyrin in a 5' or a 3' linkage,or to both types of linkages to allow the texaphyrin to be an internalresidue in the conjugate. The oligonucleotide or other site-directingmolecule may be attached either directly to the texaphyrin via a linker,or a couple of variable length. During treatment, for example, thetexaphyrin portion of a texaphyrin metal complex-oligonucleotideconjugate of the present invention is envisioned as being placed in thevicinity of the targeted tissue upon binding of the oligonucleotide toits complementary DNA or RNA.

Representative examples of useful steroids include any of the steroidhormones of the following five categories: progestins (e.g.progesterone), glucocorticoids (e.g., cortisol), mineralocorticoids(e.g., aldosterone), androgens (e.g., testosterone) and estrogens (e.g.,estradiol).

Representative examples of useful amino acids of peptides orpolypeptides include amino acids with simple aliphatic side chains(e.g., glycine, alanine, valine, leucine, and isoleucine), amino acidswith aromatic side chains (e.g., phenylalanine, tryptophan, tyrosine,and histidine), amino acids with oxygen and sulfur-containing sidechains (e.g., serine, threonine, methionine, and cysteine), amino acidswith side chains containing carboxylic acid or amide groups (e.g.,aspartic acid, glutamic acid, asparagine, and glutamine), and aminoacids with side chains containing strongly basic groups (e.g., lysineand arginine), and proline. Representative examples of useful peptidesinclude any of both naturally occurring and synthetic di-, tri-, tetra-,pentapeptides or longer peptides derived from any of the above describedamino acids (e.g., endorphin, enkephalin, epidermal growth factor,poly-L-lysine, or a hormone). Representative examples of usefulpolypeptides include both naturally occurring and synthetic polypeptides(e.g., insulin, ribonuclease, and endorphins) derived from the abovedescribed amino acids and peptides.

The term "a peptide having affinity for a biological receptor" meansthat upon contacting the peptide with the biological receptor, forexample under appropriate conditions of ionic strength, temperature, pHand the like, specific binding will occur. The interaction may occur dueto specific electrostatic, hydrophobic, entropic or other interaction ofcertain amino acid or glycolytic residues of the peptide with specificamino acid or glycolytic residues of the receptor to form a stablecomplex under the conditions effective to promote the interaction. Theinteraction may alter the three-dimensional conformation and thefunction or activity of either or both the peptide and the receptorinvolved in the interaction. A peptide having affinity for a biologicalreceptor may include an endorphin, an enkephalin, a growth factor, e.g.epidermal growth factor, poly-L-lysine, a hormone, a peptide region of aprotein and the like. A hormone may be estradiol, for example.

For the above-described texaphyrins, the couple may be an amide,disulfide, thioether or ether covalent bond; the oligonucleotide, theantibody, the hormone or the sapphyrin may have binding specificity forlocalization to a treatment site; and the biological receptor may belocalized to a treatment site.

Generally, water-soluble texaphyrins are preferred for the applicationsdescribed herein. "Water-soluble" means soluble in aqueous fluids toabout 1 mM or better. Such characteristics allow these texaphyrins to beuseful in a biological environment. Improved water solubility can beachieved by, for example, substituents chosen from saccharides orhydroxylated substituents.

A preferred embodiment of the present invention is a texaphyrin whereinwhen either R₅ or R₁₀ is other than hydrogen, then R₆ or R₉,respectively, is hydrogen, halide other than iodide (preferably fluoro),or hydroxyl.

A further preferred embodiment of the present invention is a then R₅ orR₁₀, respectively, is hydrogen or methyl.

Other preferred functionalizations are where R₆ and R₉ are hydrogen,then R₅, R₁₀, R₁₁ and R₁₂ are aryl, lower alkyl or lower hydroxyalkyl.The lower alkyl is preferably methyl or ethyl, more preferably methyl.The lower hydroxyalkyl is preferably of 1 to 6 carbons and 1 to 4hydroxy groups, more preferably 3-hydroxypropyl. The aryl is preferablyphenyl, either unsubstituted or substituted, preferably unsubstituted.

Further preferred embodiments of the present invention are where R₂ andR₃ are CH₂ CH₃ and R₄ is CH₃, where R₅ and R₁₀ are methyl, or where R₅and R₁₀ are (CH₂),CH₃ where n is 0, 1, 2, 3 or 4. Furthermore, R₅ andR₁₀ may be aryl having an R₁₃ substituent where R₁₃ is hydrogen, nitro,carboxy, sulfonic acid, hydroxy, oxyalkyl or halide. A presentlypreferred aryl is phenyl. The derivatization of the R₁₃ group may occurafter the condensation of the macrocycle. Preferred substituents for R₆include carboxy, alkyl or carboxyamidealkyl having a tertiary amidelinkage. Preferred substituents for R₇, R₈ and R₉ are oxyalkyl orhydroxyalkyl.

Further preferred texaphyrins are wherein each of R₁ -R₁₂ is any one ofthe substituents of Tables A and B described herein below; morepreferred texaphyrins are texaphyrins A1-A56 of Tables A and B describedherein below. Preferred metals are Mn(II), Mn(III), Y(III), Lu(III),La(III), In(III), Gd(III), Eu(III), and Dy(III).

Electron-donating substituents at the 2, 7, 12, 15, 18 and/or 21positions of the macrocycle stabilize the molecule against decompositionprocesses involving hydrolysis of the imine bonds. Such substituentsalso stabilize the resulting complex against demetallation bycontributing electrons to the aromatic π system. Such electron-donatinggroups include hydroxyl, alkyl, haloalkyl other than iodoalkyl, aryl,hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, saccharide, carboxyalkyl,carboxyamidealkyl, a site-directing molecule, or a couple to any ofthese molecules. Hydrolysis-resistant texaphyrin metal complexes areuseful for localization, magnetic resonance imaging, radiosensitization,radiation therapy, fluorescence imaging, photodynamic therapy andapplications requiring singlet oxygen production for cytotoxicity.

Electron-withdrawing substituents at the 15, 16, 17 and/or 18 positionsof the macrocycle destabilize the aromatic π system and render themacrocycle more readily reduced, i.e. more easily able to gain anelectron to form a radical. Such electron-withdrawing groups includehalide other than iodide, formyl, acyl, carboxy, or nitro substituents.Readily reducible texaphyrin metal complexes are useful forradiosensitization where the extent of radiation damage is dependent onthe generation of hydroxyl and texaphyrin radicals.

The photophysical properties of prior texaphyrin metal complexes arereported in U.S. Pat. No. 5,252,720 and include strong low energyoptical absorptions in the 690-880 nm spectral range, a high tripletquantum yield and efficient production of singlet oxygen. Texaphyrinmetal complexes of grandparent application Ser. No. 08/135,118,incorporated by reference herein, demonstrate enhanced cytotoxicity fromradiation and enhanced nucleic acid strand scission in the presence of agadolinium(III) metallotexaphyrin complex. U.S. Pat. No. 5,252,720describes photosensitized inactivation of enveloped viruses and magneticresonance imaging (MRI) of atheroma, liver, kidney and tumor usingvarious substituted texaphyrin metal complexes. Altering the polarityand electrical charges of side groups of the texaphyrin macrocyclesalters the degree, rate, and site(s) of binding to free envelopedviruses such as HIV-1 and to virally-infected peripheral mononuclearcells, thus modulating photosensitizer take-up and photosensitization ofleukemia or lymphoma cells contaminating bone-marrow. Powerfultechniques include the use of these texaphyrins in magnetic resonanceimaging followed by photodynamic therapy in the treatment of atheromaand benign and malignant tumors, or followed by sensitized X-raytreatment.

It is contemplated that the texaphyrins of the present invention willprove useful in a variety of applications. One example is in a method ofdeactivating a retrovirus or enveloped virus in an aqueous fluid. Such amethod comprises the step of adding a texaphyrin metal complex having asubstituent at the 2, 7, 12, 15, 18 and/or 21 position to said aqueousfluid and exposing the mixture to light to effect the formation ofsinglet oxygen.

The aqueous fluid may be a biological fluid, blood, plasma, edema tissuefluid, ex vivo fluid for injection into body cavities, cell culturemedia, or a supernatant solution from cell culture and the like.

In blood, an exemplary viral deactivating method would include: i)mixing with blood in vitro or ex vivo a texaphyrin metal complex havinga substituent at the 2, 7, 12, 15, 18 and/or 21 position capable ofproducing singlet oxygen when irradiated in the presence of oxygen; andii) photoirradiating the mixture in vitro or ex vivo to produce singletoxygen in a quantity cytotoxic to said retrovirus or enveloped virus.Exemplary retroviruses or enveloped viruses include herpes simplex virusI, cytomegalovirus, measles virus, or human immunodeficiency virusHIV-1. However, it is contemplated that the utility of the invention isnot limited to these viruses. Preferred metal cations are diamagneticmetal cations and a preferred metal complex is the Lu(III), La(III) orIn(III) complex of said texaphyrin.

A further application of the present invention is a method oflight-induced singlet oxygen production comprising subjecting atexaphyrin metal complex having a substituent at the 2, 7, 12, 15, 18and/or 21 position to light in the presence of oxygen. A method ofphotosensitization comprising the production of singlet oxygen byirradiating a texaphyrin metal complex having a substituent at the 2, 7,12, 15, 18 and/or 21 position and an absorption range from about 730 toabout 770 nanometers to form long-lived triplet states in high yield isanother embodiment of the present invention. A texaphyrin metal complexhaving a substituent at the 2, 7, 12, 15, 18 and/or 21 position has thestructure as described previously herein; however, for theseapplications, M is a diamagnetic metal cation, for example, In(III),Zn(II), Cd(II), Lu(III) or La(III). "Intrinsic biolocalizationselectivity" means having an inherently greater affinity for certaintissues relative to surrounding tissues.

Further aspects of the present invention include the use of a texaphyrinparamagnetic-metal complex having a substituent at the 2, 7, 12, 15, 18and/or 21 position in the following methods which take advantage of thehigh relaxivity of these compounds: i) a method of enhancement ofrelaxivity comprising the administration of said texaphyrin; ii) amethod of magnetic resonance image enhancement comprising administeringto a subject an effective amount of said texaphyrin; iii) a method ofdetection of neoplastic tissue in a patient comprising the steps ofadministering to a patient said texaphyrin in an amount effective toenhance a magnetic resonance image and detecting neoplastic tissue bymagnetic resonance imaging of said patient; iv) a method of imaging anorgan in a patient comprising administering to a patient said texaphyrinin an amount effective to enhance a magnetic resonance image of theorgan and detecting the organ by magnetic resonance imaging of saidpatient (the organ may be liver, kidney or the upper GI tract); v) amethod of imaging atheroma in a patient comprising administering to apatient said texaphyrin in an amount effective to enhance a magneticresonance image of atheroma and detecting atheroma by magnetic resonanceimaging of said patient.

For use in these imaging applications, the texaphyrin paramagnetic-metalcomplex has the structure as described herein; however, M is aparamagnetic metal cation, such as a trivalent lanthanide metal otherthan La(III), Lu(III) and Pm(III). In particular, M may be Mn(II),Mn(III), Fe(III) or Gd(III) and is preferably Gd(III).

A method of treating a host harboring atheroma or benign or malignanttumor cells is also an aspect of the invention. An exemplary preferredmethod includes administering to the host as a first agent a texaphyrindetectable-metal complex having a substituent at the 2, 7, 12, 15, 18and/or 21 position, said complex exhibiting selective biolocalization insuch atheroma or tumor cells relative to surrounding tissue; determininglocalization sites in the host by reference to such detectable metal;administering to the host as a second agent a texaphyrindiamagnetic-metal complex having a substituent at the 2, 7, 12, 15, 18and/or 21 position and having essentially identical biolocalizationproperty and exhibiting the ability to generate singlet oxygen uponexposure to light; and photoirradiating the second agent in proximity tosaid atheroma or tumor cells.

In the above-described method, the first agent is further defined asbeing a texaphyrin paramagnetic-metal complex, the paramagnetic metalserving as the detectable metal. In this case, determination oflocalization sites occurs by magnetic resonance imaging; and the secondagent is a texaphyrin diamagnetic-metal complex. The paramagnetic metalis most preferably Gd(III) and the diamagnetic metal is most preferablyLa(III), Lu(III) or In(III). A variation of this method uses as a firstagent a texaphyrin-gamma emitting metal complex that serves as adetectable metal, determination of localization sites occurs by gammabody scanning and the second agent is a texaphyrin-diamagnetic metalcomplex. A further variation uses as a first agent a texaphyrin whichexhibits fluorescence, e.g., a texaphyrin that is non-metallated (M=H)or is complexed with a diamagnetic metal. Localization means is then byfluorescent spectroscopy. where M is hydrogen or a detectable metal,preferably detectable by magnetic resonance imaging, by gamma scanningor fluorescence spectroscopy. "Detectable" as used herein means that thelocation may be found by localization means such as magnetic resonanceimaging if the metal is paramagnetic, gamma ray detection if the metalis gamma emitting or using monochromatic X-ray photon sources or byfluorescence. "Selective biolocalization" means having an inherentlygreater affinity for certain tissues relative to surrounding tissues."Essentially identical biolocalization property" means the second agentis a texaphyrin derivative having about the same selective targetingcharacteristics in tissue as demonstrated by the first agent.

A method of treating a host harboring tumor cells comprises the stepsof: i) administering to the host an effective amount of a texaphyrindiamagnetic-metal complex having a substituent at the 2, 7, 12, 15, 18and/or 21 position, the complex exhibiting selective biolocalization inthe tumor cells relative to surrounding tissue; and ii) photoirradiatingthe texaphyrin-diamagnetic metal complex in proximity to the tumorcells. The photoirradiating is generally at a wavelength of about 730 to770 nanometers or may be from laser light. In these embodiments, thediamagnetic metal will typically be In(III), La(III) or Lu(III).

The present invention provides a method of radiation therapy for a hostharboring a tumor. The method includes the steps of administering to thehost a texaphyrin having a substituent in the 2, 7, 12, 15, 18 and/or 21position(s), and administering ionizing radiation to the host inproximity to the tumor either before or after administration of thetexaphyrin, following procedures as described in U.S. Ser. No.08/135,118, incorporated herein by reference. The texaphyrin exhibitsgreater biolocalization in the tumor relative to non-tumor tissue andhas radiosensitization properties. A tumor may be a benign or malignanttumor or may be atheroma. A texaphyrin having radiosensitizationproperties enhances cytotoxicity from ionizing radiation as compared tocontrol experiments without the texaphyrin. Ionizing radiation includesbut is not limited to x-rays, and internal and external gamma emittingradioisotopes.

The texaphyrin may be complexed with a metal; however, a metal is notnecessary for radiosensitization. The metal is important to thestability of the texaphyrin complex. The ionizing radiation may be froman external source or the metal may be a radioactive metal. In thelatter case, the ionizing radiation is from the radioactive metal incombination with radiation from an external source.

An improved method of treating a host harboring a tumor comprises thefurther step of determining localization sites in the host by monitoringtexaphyrin concentrations. "Monitoring texaphyrin concentrations" meansmeasuring fluorescence of an administered free base texaphyrin or byreference to the metal of an administered texaphyrin metal complex. Ifthe metal is paramagnetic, then magnetic resonance imaging is used formeasurement; if the metal is a gamma emitting radioactive metal, thengamma emission is used for measurement.

A further improved method of treating a host harboring a tumor comprisesthe additional steps of administering to the host as a second agent atexaphyrin-diamagnetic metal complex having a substituent at the 2, 7,12, 15, 18 and/or 21 position and having essentially identicalbiolocalization property and administering ionizing radiation andphotoirradiation in proximity to the tumor.

In these methods, determining localization sites may occur by observingfluorescence from the texaphyrin. When the first agent is complexed witha metal, the metal may be a gamma-emitting metal and determininglocalization sites would occur by gamma body imaging, or the metal maybe a paramagnetic metal and determining localization sites would occurby magnetic resonance imaging. "Exhibiting greater biolocalization inthe tumor relative to non-tumor tissue" means having an inherentlygreater affinity for tumor tissue relative to non-tumor tissue. Thesecond agent has essentially identical biolocalization property as thefirst agent and exhibits the ability to generate singlet oxygen uponexposure to light. The photodynamic effect may be derived from anaerobicelectron transfer processes. A preferred diamagnetic metal texaphyrincomplex is the Lu(III), La(III) or In(III) complex of a texaphyrin."Essentially identical biolocalization property" means the second agentis a texaphyrin derivative having about the same selective targetingcharacteristics in tissue as demonstrated by the first agent. The firstagent and the second agent may be the same texaphyrin.

A preferred embodiment of the present invention is a method of radiationtherapy for a host harboring a tumor comprising the steps of i)administering to the host a pharmaceutically effective amount of the Gdcomplex of a texaphyrin having a substituent at the 2, 7, 12, 15, 18and/or 21 position(s); and ii) administering ionizing radiation to thehost in proximity to the tumor, either before or after administration ofthe texaphyrin metal complex.

Another aspect of this invention is a method of imaging atheroma in ahost comprising the administration to the host as an agent adetectable-metal texaphyrin complex having a substituent at the 2, 7,12, 15, 18 and/or 21 position(s), said complex exhibiting selectivebiolocalization in such atheroma; and imaging the atheroma in the hostby reference to such detectable metal. The agent is preferably adetectable-metal texaphyrin complex having a paramagnetic metal servingas said detectable metal, and imaging of the atheroma occurs by magneticresonance imaging. The paramagnetic metal is preferably Gd(III).

For the above-described uses, texaphyrins are provided as pharmaceuticalpreparations. A pharmaceutical preparation of a texaphyrin may beadministered alone or in combination with pharmaceutically acceptablecarriers, in either single or multiple doses. Suitable pharmaceuticalcarriers include inert solid diluents or fillers, sterile aqueoussolution and various organic solvents. The pharmaceutical compositionsformed by combining a texaphyrin of the present invention and thepharmaceutically acceptable carriers are then easily administered in avariety of dosage forms such as injectable solutions.

For parenteral administration, solutions of the texaphyrin in sesame orpeanut oil, aqueous propylene glycol, or in sterile aqueous solution maybe employed. Such aqueous solutions should be suitably buffered ifnecessary and the liquid diluent first rendered isotonic using, forexample, saline or glucose. These particular aqueous solutions areespecially suitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy use with a syringe exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars such as mannitol or dextrose or sodiumchloride. A more preferable isotonic agent is a mannitol solution ofabout 2-8% concentration, and, most preferably, of about 5%concentration. Prolonged absorption of the injectable compositions canbe brought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The following examples describe the synthesis of texaphyrin metalcomplexes having a substituent(s) at the 2, 7, 12, 15, 18 and/or 21position(s) of the macrocycle. Unless defined otherwise, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, the preferred methods and materials are nowdescribed. Unless mentioned otherwise, the techniques employed hereinare standard methodologies well known to one of ordinary skill in theart.

EXAMPLE 1 Synthesis of Compounds A3, A5, A6 and A7

This example describes the synthesis of a texaphyrin metal complexhaving substituents at the 12 (R₅), 15 (R₆), 18 (R₉) and 21 (R₁₀)positions of the macrocycle as depicted in Scheme A, parts 1 and 2; atripyrrane ketone A5, a substituted ortho-phenylenediamine A3, anonaromatic texaphyrin A6, and a metal complex of aromatic texaphyrinA7.

All solvents and reagents are of reagent grade quality, availablecommercially, and are used without further purification. Sigmalipophilic Sephadex (LH-20-100) and Merck type 60 (230-400 mesh) silicagel are used for column chromatography.

¹ H and ¹³ C NMR spectra are obtained on a General Electric QE-300 (300MHz.) spectrometer. Electronic spectra are recorded on a Beckman DU-7spectrophotometer in CHCl₃. Infrared spectra are recorded, as KBrpellets, from 4000 to 600 cm⁻¹ on a Nicolet 510P FT-IRspectrophotometer. Chemical ionization mass spectrometric analyses (CIMS) are made using a Finnigan MAT 4023. Low resolution and highresolution fast atom bombardment mass spectrometry (FAB MS) areperformed with a Finnigan-MAT TSQ-70 and VG ZAB2E instruments,respectively. A nitrobenzyl alcohol (NBA) matrix is utilized with CHCl₃as the co-solvent. Elemental analyses are performed by AtlanticMicrolab, Inc. Melting points are measured on a Mel-temp apparatus andare uncorrected. ##STR4##

Tripyrrane ketone AS: An example of the synthesis of a precursor to atripyrrane ketone, the 2,5-bis(3-(3-hydroxypropyl)-5-carboxyl-4-methylpyrrol-2-yl)methyl!-3,4-diethylpyrroleF5, Scheme F, was presented in prior application, U.S. Ser. No.08/135,118, incorporated by reference herein. In this example, R₁ is3-hydroxypropyl, R₂ and R₃ are ethyl and R₄ is methyl.

The synthesis of compound F5 provides teachings for the synthesis of A4,precursor to tripyrrane ketone A5 as shown in Scheme F and describedherein. ##STR5##

2,5-Bis(5-benzyloxycarbonyl-4-methyl-3-methoxycarbonylethylpyrrol-2-yl)methyl!-3,4-diethylpyrrole.F3, Scheme F. In a 500 mL round bottom flask was placed 250 mL ofethanol from an unopened bottle which is purged with dry nitrogen forten minutes. 3,4-Diethylpyrrole F2 (1.29 g, 0.01 mol) and2-acetoxymethyl-5-benzyloxycarbonyl-4-methyl-3-methoxycarbonylethylpyrroleF1 (7.83 g, 0.02 mol) were added and the mixture heated until all of thepyrroles dissolved. p-Toluenesulfonic acid (65 mg) was added and thereaction temperature maintained at 60° C. The reaction slowly changedcolor from a clear yellow to a dark red with the product precipitatingout of the solution as the reaction progressed. After ten hours thereaction was cooled to room temperature, the volume reduced to one halfon a rotary evaporator, and then placed in the freezer for severalhours. The product was collected by filtration, washed with a smallamount of cold ethanol to afford 4.61 g of an off-white fine powder(61%): ¹ H NMR (CDCl₃, 250 MHz): δ 1.14 (6H, t, CH₂ CH₃), 2.23 (6H, s,pyrrole--CH₃), 2.31 (4H, t, CH₂ CH₂ CO₂ CH₃), 2.50 (4H, q, CH₂ CH₃),2.64 (4H, t, CH₂ CH₂ CO₂ CH₃), 3.60 (10H, br s, CH₃ CO₂ -- and(pyrrole)₂ --CH₂), 4.44 (4H, br s, C₆ H₅ CH₂), 6.99-7.02 (4H, m,aromatic), 7.22-7.26 (6H, m, aromatic), 8.72 (1H, s, NH), 10.88 (2H, brs, NH); ¹³ C NMR (CDCl₃, 250 MHz): δ 10.97, 16.78, 17.71, 19.40, 22.07,35.09, 51.46, 65.32, 117.37, 119.34, 122.14, 126.58, 126.79, 127.36,128.19, 133.55, 136.62, 162.35, 173.49; CI MS (M+H)+750; HRMS 749.3676(calc. for C₄₄ H₅₁ N₃ O₈ : 749.3676).

A synthetic scheme is presented in Scheme G for the attachment of anester, a carboxyl and a tertiary amide as R₂ and R₃ substituents. Thesynthesis of compound G1 is described in Kaesler et al. (1983). ##STR6##

2,5-Bis(5-benzyloxycarbonyl-3-(3-hydroxypropyl)-4-methylpyrrol-2-yl)methyl!-3,4-diethylpyrrole.F4, Scheme F. 2,5-Bis(5-benzyloxycarbonyl-4-methyl-3-methoxycarbonylethylpyrrol-2-yl)methyl!-3,4-diethylpyrroleF3 (5.00 g, 0.007 mol) was placed in a three necked 100 mL round bottomflask and vacuum dried for at least 30 minutes. The flask was equippedwith a thermometer, an addition funnel, a nitrogen inlet tube, and amagnetic stir bar. After the tripyrrane was partially dissolved into 10mL of dry THF, 29 mL of borane (1M BH₃ in THF) was added dropwise withstirring. The reaction became mildly exothermic and was cooled with acool water bath. The tripyrrane slowly dissolved to form a homogeneousorange solution which turned to a bright fluorescent orange color as thereaction went to completion. After stirring the reaction for one hour atroom temperature, the reaction was quenched by adding methanol dropwiseuntil the vigorous effervescence ceased. The solvents were removed underreduced pressure and the resulting white solid redissolved into CH₂ Cl₂.The tripyrrane was washed three times with 0.5M HCl (200 mL total),dried over anhydrous K₂ CO₃, filtered, and the CH₂ Cl₂ removed underreduced pressure until crystals of the tripyrrane just started to form.Hexanes (50 Ml) was added and the tripyrrane allowed to crystallize inthe freezer for several hours. The product was filtered and againrecrystallized from CH₂ Cl₂ /ethanol. The product was collected byfiltration and vacuum dried to yield 3.69 g of an orangish white solid(76%): mp 172°-173° C.; ¹ H NMR (CDCl₃, 300 MHz): δ 1.11 (6H, t, CH₂CH₃), 1.57 (4H, p, CH₂ CH₂ CH₂ OH), 2.23 (6H, s, pyrrole--CH₃),2.39-2.49 (8H, m, CH₂ CH₃ and CH₂ CH₂ CH₂ OH), 3.50 (4H, t, CH₂ CH₂ CH₂OH), 3.66 (4H; s, (pyrrole)₂ --CH₂), 4.83 (4H, s, C₆ H₅ --CH₂),7.17-7.20 (4H, m, aromatic), 7.25-7.30 (6H, m, aromatic), 8.64 (1H, s,NH), 9.92 (2H, s, NH); ¹³ C NMR (CDCl₃, 300 MHz): δ 10.97, 16.72, 17.68,20.00, 22.38, 33.22, 62.01, 65.43, 117.20, 119.75, 120.72, 122.24,127.23, 127.62, 128.30, 132.95, 136.60, 162.13; FAB MS (M+) 693.

2,5-Bis (3-(3-hydroxypropyl)-5-carboxyl-4-methyl pyrrol-2-yl)methyl!-3,4-diethylpyrrole F5, Scheme F. 2,5-Bis(3-(3-hydroxypropyl)-5-benzyloxycarbonyl-4-methylpyrrol-2-yl)methyl!-3,4-diethylpyrroleF4 (15.0 g, 0.02 mol) was placed in a 1 L round bottom flask and driedin vacuo for ca. 30 min. The tripyrrane was dissolved in dry THF (600mL) with triethylamine (10 drops) and 10% Pd on carbon (600 mg) and thereaction was stirred at room temperature under one atmosphere of H₂.After 15 h, the suspension was filtered through celite to remove thecatalyst and the resulting clear solution was concentrated under reducedpressure to yield a light pink solid. This material, obtained in nearquantitative yield, was taken on to the next step without furtherpurification.

A carboxyl tripyrrane A4 (a specific example presented as F5 in SchemeF) (0.02 mol) is placed in a 250 mL round bottom flask and dried invacuo for ca. 1 h. At room temperature under nitrogen, trifluoroaceticacid (31 mL, 0.40 mol) is added dropwise via syringe. The tripyrranedissolves with visible evolution of CO₂ to form a homogeneous yellowsolution. The reaction is stirred at room temperature for ca. 15 min,then cooled to 0° C. using a water/ice bath. A triethyl-ortho-ester (ortrimethyl-ortho-ester, ca. 18 eq) is added to the reaction mixturedropwise with stirring after which the reaction is stirred for anadditional 15 minutes at 0° C. If the ester is acetate, then a methylgroup would be attached, propionate would attach an ethyl group, forexample. The reaction is warmed to room temperature and 100 mL of wateradded dropwise. After stirring the resulting two phase mixture for ca.30 minutes, the reaction mixture is extracted three times with CH₂ Cl₂.The CH₂ Cl₂ extracts are combined and washed three times with 1M aq.NaHCO₃, once with water, dried over anhydrous sodium sulfate, filtered,and the solvent removed under reduced pressure. The resulting solid isrecrystallized from CH₂ Cl₂ /hexanes.

Substituted ortho-phenylenediamine: The synthesis of anortho-phenylenediamine substituted at the 4 and 5 positions is describedin U.S. Pat. No. 5,252,720 and application Ser. No. 08/135,118.

Texaphyrin macrocycles having a free carboxyl or a free amino group forfurther derivatization on the benzene ring portion of the molecule maybe synthesized by replacing ortho-phenylenediamine with3,4-diaminobenzoic acid or 3,4-diaminoaniline. One skilled in the art oforganic synthesis would realize in light of the present disclosure thatother substituted 1,2-o-phenylenediamines may be used as a precursor,e.g., a 1-2-o-phenylenediamine that is differentially substituted in the4 and 5 positions. This substitution may be the result of differentfunctionalities being present or specific protection and standardorganic and/or biochemical transformations having been carried out. Suchmacrocycles can be further functionalized to derivatives having anantibody, oligonucleotide, protein, peptide, sapphyrin and the like onone position of the B portion of the molecule.

Synthesis of A3, Scheme A: Compound A1 of Scheme A (a1,2-dialkyl-4,5-dinitrobenzene) is reacted with an alkyl halide wherethe halide is chloride, bromide or iodide in the presence of a Lewisacid such as AlCl₃, for example. The 3 and 6 positions of the phenylring are derivatized with the alkyl group to form compound A2. A mixtureof reactants having a single halide and different alkyl groups may beused to generate different alkyl derivatives at the 3 and 6 positions.The yield of a particular product would be lower in this case.

A diamine A3 (Scheme A) is obtained by reduction of the correspondingsubstituted dinitrobenzene (A2, Scheme A) with hydrazine hydrate (1 mL)and 10% palladium on carbon (50 mg) in 40 mL refluxing absolutemethanol. The resulting suspension may bubble for approximately 15-20minutes and then turn colorless after 1 hour. At this point thereduction is complete as verified by TLC. The reaction solution is hotfiltered through celite into a dry flask, covered with aluminum foil,and then concentrated to an oil. The diamine is taken to the next stepwithout further purification. Ammonium formate in the presence ofpalladium (10% on carbon) catalyst may act as a mild, inexpensive andsafe alternative to hydrazine hydrate in the above reaction and would beused, for example, when sensitive groups such as amide are present atother positions of the molecule.

Condensation of a tripyrrane ketone and a substitutedortho-phenylenediamine to form a nonaromatic texaphyrin havingsubstituents at the 2, 7, 12, 15, 18 and/or 21 position(s): A tripyrraneketone and a substituted ortho-phenylenediamine having substituents atthe 3 and/or 6 position(s) are placed in a 2 L round bottom flask with1000 mL of toluene and 200 mL of methanol. The solvents are purged withnitrogen prior to use. Concentrated HCl (0.5 mL) is added and thereaction heated to reflux under nitrogen. After 5 h the reaction iscooled to room temperature and the solvents removed under reducedpressure until the product precipitates out of solution. The remainderof the solvent is decanted off and the macrocycle is dried in vacuo. Theproduct is recrystallized from methanol/diethylether and characterizedby ¹ H NMR and ¹³ C NMR.

Condensation of a diformyltripyrrole and a substitutedortho-phenylenediamine yields a nonaromatic texaphyrin havingsubstituents in the 15, 16, 17 or 18 positions.

General procedure for the synthesis of a metal complex of texaphyrin(A7, Scheme A). One equivalent of the hydrochloride salt of themacrocycle A6, 1.5 equivalents of the M(OAc₃)₃ ·XH₂ O metal salt (whereM=metal ion),and triethylamine (ca. 1 mL) are mixed together in methanoland heated to reflux under air. After completion of the reaction (asjudged by the UV/vis spectrum of the reaction mixture), the solution iscooled to room temperature, the solvent is removed under reducedpressure and the crude complex dried in vacuo for several hours. Asolution of dichloromethane/methanol (99:1 v/v) is added to the crudecomplex and the suspension is sonicated a few min. The suspension isfiltered in order to remove impurities in the filtrate (incompleteoxidation products and excess triethylamine). The resulting solid isdissolved in methanol and then chloroform is added to reduce thepolarity of the mixture (1:2 v/v). This solution is filtered throughcelite and loaded on a (pre-treated/pre-washed 1M NaNO₃) neutral aluminacolumn (10 cm). The column is first eluted with a 1:10 (v/v)methanol/chloroform solution by gravity to remove any impurity. Themetal complex is then obtained by eluting the column with chloroformcontaining increasing amounts of methanol (20-50%). The purifiedlanthanide(III) texaphyrin complex is recrystallized by dissolving thecomplex in methanol/chloroform and carefully layering the solution witha small amount of methanol, then with diethylether. The layered solutionis kept at room temperature in the dark for a few days. The texaphyrinmetal complex is recrystallized twice for analytically pure measurementsand characterizations.

Alternatively, the crude metal complex may be isolated by mixing thecomplex with an aqueous solution of a salt at a temperature above thefreezing point of the resulting mixture, and then recovering theprecipitated texaphyrin from the mixture. The salt can be any salt thatis soluble in water or a water/organic solvent mixture and does notcause transmetallation of the texaphyrin metal complex.

The texaphyrin metal complex may alternatively be purified by dissolvingthe complex in water and methanol, and acid-washed zeolites (such asLZY-54 zeolite) are then added to the solution. The mixture is agitatedfor a period of time and is then filtered to remove the zeolites. Thisprocedure may be repeated 2 or more times until significantly all of thefree metal ion is removed.

Lanthanum(III), Cerium(III), Praseodymium(III), Neodymium(III),Samarium(III), Europium(III), Gadolinium (III), Terbium(III),Dysprosium(III), Holmium(III), Erbium(III), Thulium(III),Ytterbium(III), Lutetium(III) complexes of texaphyrin: The hydrochloridesalt of macrocycle A6 (0.407 mmol), and one of the following lanthanidesalts: La(OAc₃)₃ ·6H₂ O (0.814 mmol), Ce(OAc₃)₃ ·6H₂ O (0.611 mmol),Pr(OAc₃)₃ ·5H₂ O (0.611 mmol), Nd(OAc₃)₃ ·6H₂ O(0.611 mmol), Sm(OAc₃)₃·5H₂ O (0.611 mmol), Eu(OAc₃)₃ ·5H₂ O (0.65 mmol), Gd(OAc₃)₃ ·5H₂ O (1.5mmol), Tb(OAc₃)₃ ·6H₂ O (0.611 mmol), Dy(OAc₃)₃ ·5H₂ O (0.611 mmol),Ho(OAC3)₃ ·5H₂ O (0.611 mmol), Er(OAc₃)₃ ·5H₂ O (0.611 mmol), Tm(OAc₃)₃·5H₂ O (0.611 mmol), Yb(OAc₃)₃ ·5H₂ O (0.611 mmol), or Lu(OAc₃)₃ ·H₂ O(0.611 mmol), together with TBANO₃ (1.0 mmol) and triethylamine (ca. 0.5mL) in 350 mL methanol are heated to reflux under air for 5-24 h. Theworkup uses the general procedure outlined above. The thulium andlutetium complexes may be more difficult to purify due to their lowersolubility in methanol/chloroform solutions, which leads to a loweryield.

EXAMPLE 2 Synthesis of Compounds B4, C5 and D5

Ortho-phenylenediamine compounds having substituents bound to the phenylring via an oxygen are prepared as indicated in Schemes B and C.##STR7##

2,3,4-Trihydroxybenzoic acid B1, is reacted with an alkyl 5 halide wherethe halide is chloride, bromide, or iodide in the presence of potassiumcarbonate and acetonitrile to form a trialkoxy derivative B2. The alkylgroup of the halide may be a primary or secondary alkyl having one ormore hydroxy, alkoxy, carboxy, ester, amine, amide or protected aminesubstituents at positions at least one carbon removed from the site ofhalide attachment. These alkyl groups may be unsubstituted, singly ormultiply functionalized. They may be branched or unbranched. Preferredalkyl groups are methyl, hydroxypropyl or methoxy(ethoxy)nethoxy(n=1-100; a polyethylene glycol substituent). Compound B2 is reactedwith 90% nitric acid to form the dinitro derivative B3 which is thenreacted with either hydrazine hydrate or ammonium formate and 10%palladium on carbon in methanol to form compound B4.

In a similar synthesis, starting with 2,3,4-trihydroxybenzaldehyde C1(Scheme C), reduction of the trialkoxy derivative C2 with hydrazine inKOH results in a methyl derivative at the R₆ position to form1,2,3-trialkoxy-4-methylbenzene C3. The diamine is formed as depicted inScheme B and described above.

Scheme D shows the formation of a tertiary amine at the R₆ position. Thestarting material is 2,3,4-trihydroxybenzoic acid (D1). Compound D3 (B3)is treated with an amine component in 1,3-dicyclohexylcarbodiimide anddimethylformamide to form D4 having an amide linkage. Alternativecoupling reagents include 1,1-carbonyldiimidazole (CDI) or ECC.Reduction as described above yields the diamine for condensation with atripyrrane ketone. ##STR8##

EXAMPLE 3 Synthesis of a T2B4 Texaphyrin

Scheme E, parts 1 and 2, shows the synthesis of a lanthanide metalcomplex of a T2B4 texaphyrin. A diformyltripyrrole E5 is condensed witha substituted ortho-phenylenediamine E4 to form the nonaromaticprecursor E6. The synthesis of the substituted ortho-phenylenediamine E4was described in example 2 and the diformyltripyrrole was described inU.S. Pat. No. 5,252,720. In this example, R' may be polyethylene glycol(PEG) where the number of repeating ethoxy units may be as many as 200,a saccharide, a polyhydroxy substituent or the like. R may be methoxy,methyl or hydrogen. ##STR9##

EXAMPLE 4 Synthesis of a Tripyrrane Having Meso-Substituents

Scheme A, parts 1 and 2, refers to the structure of a metallotexaphyrinwith substituents in the 2 and 7 positions (meso-positions). Texaphyrinmacrocycles having meso-substitution on the periphery of the aromaticmacrocycle may be synthesized by first preparing newmethylene-functionalized tyripyrrane dialdehydes described in Scheme I,parts 1 and 2. One skilled in the art of organic synthesis would realizein light of the present disclosure that a variety of1,2-o-phenylenediamines may be used to react with these newfunctionalized tripyrranes. The organic synthesis required for thevarious transformations illustrated in Scheme I is derived from classicpyrrole/porphyrin chemistry.

Synthesis of I3, Scheme I, part 1: Pyrrole I1(readily available fromAldrich Chemical Co., Milwaukee, Wis.) of Scheme I is reacted withsulfuryl chloride in dichloromethane, followed by hydrolysis with sodiumacetate, and acidification to afford the acid pyrrole, 12 (see A. R.Battersby et al., J. C. S. Perkin I, 1976, 1008). Decarboxylation viatrifluoroacetic acid yields 13 (see M. J. Cyr, Ph.D. Dissertation,University of Texas at Austin, 1992). ##STR10##

Synthesis of I5. The acid-catalyzed condensation between compound I3 andthe t-butylester derived pyrrole I4 (pyrrole I4 is described in D. H. R.Barton and S. Zard, J. C. S. Chem. Commun., 1985, 1098-1100), in thepresence of an aldehyde (R₁₂ =alkyl, aryl, etc.) will afford a mixtureof three dipyrromethanes. The desired mixed-ester derived dipyrromethaneI5 is obtained by column chromatography. The preparation ofdipyrromethanes is well-documented in the literature (see, Sessler etal., J. Org. Chem., 1986, 51, 2838).

Synthesis of I7. The t-butylester of compound I5 is selectivelydeprotected and decarboxylated via trifluoroacetic acid and subsequentlycondensed via acid-catalysis with pyrrole I3 in the presence of analdehyde (R₁₁ =alkyl, aryl, etc.) to afford the desired tripyrrane I7.

Synthesis of the diformyl tripyrrane I9. With compound I7 in hand, thetripyrrane is transformed to the desired diformyl tripyrrane I9 (R₅ =H)by standard organic synthesis reported earlier (U.S. Pat. No.5,252,720). Compound I7 is reduced by borane/THF, followed byacetylation via acetic anhydride or acetyl chloride to afford tripyrraneI8. At this point, debenzylation of I8, followed by subsequent Clezyformylation of the intermediate, and basic hydrolysis with lithiumhydroxide, provides tripyrrane I9.

Tripyrrane I9 may then be condensed with an ortho-phenylenediamine toconstruct a texaphyrin macrocycle as depicted in Scheme A. Substituentsin these meso-positions are expected to further stabilize themacrocycle.

EXAMPLE 5

2,5-Bis(3-acetoxypropyl-5-benzoyl-4-methylpyrrol-2-yl)methyl!-3,4-diethylpyrrole(J2). 2,5-Bis(3-acetoxypropyl-5-carboxyl-4-methylpyrrol-2-yl)methyl!-3,4-diethylpyrroleJ1(1.00 g, 1.67 mmol) was placed in a 100 mL three-neck round-bottomflask and dried under high vacuum for ca. 1 hr. The round-bottom flaskwas equipped with an argon inlet line and for magnetic stirring. At roomtemperature under argon, CH₂ Cl₂ (10 mL) was added to the flask and theresulting mixture stirred to form a suspension. Trifluoroacetic acid(2.7 mL) was then added all at once to the suspension. The tripyrranedissolved to form a light orange solution. The reaction was stirred atroom temperature under argon for ca. 45 min, after which it was cooledto 0° C. using an ice/water bath. Triethylorthobenzoate (3.8 mL) wasadded dropwise to the reaction with stirring over a two minute periodunder a flow of argon. The reaction was stirred for 40 min at 0° C. thenallowed to warm to room temperature over 20 min. Water (20 mL) was addedto the reaction and stirring continued for another 2 hr. Transferredreaction to a separatory funnel, separated and discarded the upperaqueous phase, and basified the lower organic layer with sat. aqueousNaHCO₃ (Caution: gas evolution and frothing occurs). Separated the twolayers and washed the organic phase once with sat. aqueous NaHCO₃ andonce with water. Dried organic phase over anhydrous MgSO₄, filtered offthe drying agent, removed the solvent under reduced pressure, and driedthe resulting orange-red oil under high vacuum overnight. The oil wasdissolved into a minimum amount of CH₂ Cl₂ (5-10 mL), the solutionlayered with hexanes (ca. 50 mL), and the tripyrrane allowed tocrystallize at -20° C. The product was collected by filtration and driedunder high vacuum to yield 1.06 grams of a tan solid (J2) (88%). ¹ H NMR(CDCl3, 300 MHz): δ 1.06 (6H, t, CH₂ CH₃), 1.67 (4H, p, CH₂ CH₂ CH₂OAc), 1.81 (6H, s, CH₃ CO₂ --), 2.02 (6H, s, pyrr--CH₃), 2.37-2.44 (8H,m, CH₂ CH₃ and CH₂ CH₂ CH₂ OAc), 3.71 (4H, s, (pyrr)₂ --CH₂), 3.99 (4H,t, CH₂ CH₂ CH₂ OAc), 7.29-7.48 (10H, m, aromatic), 9.16 (1H, s, NH),9.66 (2H, s, NH); ¹³ C NMR (CDCl₃): δ 11.8, 16.4, 17.7, 20.1, 20.9,22.7, 29.1, 63.9, 120.9, 121.5, 127.3, 128.1, 128.2, 129.1, 130.8,135.6, 140.1, 171.3, 185.7; FAB MS, M⁺ : m/e 717.

4,5-Diethyl-9,24-bis(3-hydroxypropyl)-16,17-dimethoxy-10,23-dimethyl-12,21-diphenyl-13,20,25,26,27-pentaazapentacyclo- 20.2.1.1³,6.1⁸,11.0¹⁴,19 !heptacosa-3,5,8,10,12,14,16,18,20,22,24-undecaene (J6).

2,5-Bis(3-acetoxypropyl-5-benzoyl-4-methylpyrrol-2-yl)methyl!-3,4-diethylpyrroleJ2 (100 mg, 0.14 mmol) and 4,5-dimethoxy-1,2-phenylenediamine J5 (23 mg,0.14 mmol) were dissolved into 100 mL of absolute methanol under argon.Concentrated HCl (5 drops) was added and the reaction heated at refluxunder argon. After heating for 2 days, the reaction was cooled to roomtemperature and the solvent removed under reduced pressure. Theresulting red solid was dissolved into CH₂ Cl₂ (5 mL), filtered, and theCH₂ Cl₂ solution layered with hexanes (20 mL). The product was allowedto slowly precipitate out of solution at room temperature overnight. Themother liquor was decanted off and the remaining solid washed withhexanes. After drying the solid under high vacuum, 39 mg of dark redproduct (J6) was obtained. FAB MS, (M+H)⁺ : m/e 766.

Cadmium(II) complex of4,5-diethyl-9,24-bis(3-hydroxypropyl)-16,17-dimethoxy-10,23-dimethyl-12,21-diphenyl-13,20,25,26,27-pentaazapentacyclo 20.2.1.1³,6.1⁸,11.0¹⁴,19!-heptacosa-1,3,5,7,9,11(27),12,14(19),15, 17,20,22(25),23-tridecaene(J8). The protonated form of the macrocycle J6 (11 mg, 0.014 mmol),cadmium(II) chloride (11 mg, 0.06 mmol) and triethylamine (20 mL) in 20mL of methanol were heated at reflux under air for 2 days. The reactionwas cooled to room temperature, the solvent removed under reducedpressure, and the complex dried in vacuo overnight to give the finaltexaphyrin-Cd(II) metal complex (J8). UV/vis (CH₃ OH) λ_(max), nm!:472.0, 756.0; FAB MS, (M+H)⁺ : m/e 875. ##STR11##

EXAMPLE 6

2,5-bis(5-benzoyl-3-ethyl-4-methylpyrrol-2-yl)meth-yl!-3,4-diethylpyrrole (J4).2,5-Bis(5-carboxyl-3-ethyl-4-methylpyrrol-2-yl)methyl!-3,4-diethylpyrrole J3(1.00 g, 2.20 mmol) was placed in a 100 mL three-neck round-bottom flaskand dried under high vacuum for 1 hr. The round-bottom flask wasequipped with an argon inlet line and for magnetic stirring. At roomtemperature under argon, CH₂ Cl₂ (10 mL) was added to the reaction flaskand the resulting mixture stirred to form a suspension. Trifluoroaceticacid (3.5 mL) was then added all at once to the suspension. Thetripyrrane dissolved to form a yellowish orange solution. The reactionwas stirred at room temperature under argon for ca. 35 min, after whichit was cooled to 0° C. using an ice/water bath. Triethylorthobenzoate(5.0 mL) was added dropwise to the reaction with stirring over a twominute period under a flow of argon. The reaction was stirred for 40 minat 0° C. then allowed to warm to room temperature over 20 min. Water (20mL) was added to the reaction and stirring continued for another 1 hr.Transferred reaction to a separatory funnel, separated and discarded theupper aqueous phase, and basified the lower organic layer with sat.aqueous NaHCO₃ (30 mL) (Caution: gas evolution and frothing occurs).Separated the two layers and washed the organic phase once with sat.aqueous NaHCO₃ and once with water. Dried organic phase over anhydrousMgSO4, filtered off the drying agent, and removed the solvent underreduced pressure to yield a dark oil with some precipitate. The oil andsolid were dissolved into a minimum amount of CH₂ Cl₂ (5 mL), thesolution layered with hexanes (ca. 50 mL), and the product allowed tocrystallize at -20° C. The product was collected by filtration, washedwith a small amount of hexanes, and dried under high vacuum to yield0.88 grams of a tan solid (J4) (70%). ¹ H NMR (CDCl₃, 300 MHz): δ 0.95(6H, t, CH₂ CH₃), 1.05 (6H, t, CH₂ CH₃), 1.80 (6H, s, pyrr--CH₃),2.32-2.40 (8H, m, CH₂ CH₃), 3.67 (4H, s, (pyrr)₂ --CH₂), 7.27-7.48 (10H,m, aromatic), 9.27 (1H, s, NH), 9.66 (2H, S, NH); ¹³ C NMR (CDCl₃): δ11.7, 15.1, 16.3, 17.1, 17.7, 22.8, 120.8, 121.4, 124.8, 127.0,128.0(6), 128.1(4), 129.2, 130.6, 135.5, 140.2, 185.7; FAB MS, (M+H)⁺ :m/e 574.

4,5,9,24-Tetraethyl-16,17-dimethoxy-10,23-dimethyl-12,21-diphenyl-13,20,25,26,27-pentaazapentacyclo- 20.2.1.1³,6.1⁸,11.0¹⁴,19!heptacosa-3,5,8,10,12,14,16,18,20,22,24-undecaene (J7). 2,5-Bis(5-benzoyl-3-ethyl-4-methylpyrrol-2-yl)methyl!-3,4-diethylpyrrole J4(101 mg, 0.18 mmol) and 4,5-dimethoxy-1,2-phenylenediamine J5 (30 mg,0.18 mmol) were dissolved into 200 mL of toluene and 100 mL of absolutemethanol. The solvents were sparged with argon for approximately 5 minbefore the reaction was started. Concentrated HCl (3 drops) was thenadded and the reaction heated at reflux under an atmosphere of argon.After heating for ca. 2.75 days, the reaction was cooled to roomtemperature, the solvent removed under reduced pressure and theremaining solid dried in vacuo. The macrocycle was dissolved into CH₂Cl₂ (10 mL), filtered, and the CH₂ Cl₂ solution layered with hexanes (80mL). The product was allowed to slowly precipitate out of solution at-20° C. overnight. The macrocycle was collected by filtration, dissolvedinto a minimum amount of ethanol, and the solution layered with hexanes.The macrocycle was allowed to slowly precipitate out of solution at -20°C. for several days. The macrocycle was collected by filtration, washedwith a small amount of hexanes, and dried under high vacuum to yield 28mg of dark red product (J7). FAB MS, (M+H)⁺ : m/e 707.

Macrocycle J7 can be oxidized and metallated to give the correspondingtexaphyrin metal complex following the procedures previously describedherein. ##STR12##

EXAMPLE 7 R₅, R₆, R₉, and/or R₁₀ Substituents

Scheme H, parts 1 and 2, shows a synthetic scheme for attaching a nitrogroup at position R₆ or R₉. ##STR13##

A 1,2-dialkyl-4,5-dinitrobenzene (H1, also A1) is reduced with ammoniumformate to the diamino derivative and an amine protecting group isattached before the nitration step. Amine protecting groups includeamides such as N-acetyl, and carbamates such as CBZ, for example. Anacetyl protecting group is later removed by refluxing in HCl. Protectionand deprotection procedures are well known to those of skill in the artin light of the present disclosure (Greene et al. 1991). The deprotectednitro derivative H5 is condensed with a diformyltripyrrane H6 to form anonaromatic texaphyrin having a nitro group at the 15 position.

A bromine is introduced at the R₆ and R₉ positions of the macrocycle byreacting 1,2-dialkyl-4,5-dinitrobenzene with bromine in the presence ofFeBr₃ or AlBr₃. The 3 and 6 positions of the phenyl ring are derivatizedwith bromide and reduction to the amine as described in example 2prepares the precursor for condensation with a diformyltripyrrole or atripyrrane ketone.

Preferred texaphyrins having a substituent on the 2, 7, 12, 15, 18and/or 21 position of the macrocycle are listed in Tables A and B.Substituents R₁ -R₆ are provided in Table A and R₇ -R₁₂ are provided inTable B for a given texaphyrin ("TXP").

                                      TABLE A                                     __________________________________________________________________________    Representative Substituents for Texaphyrin Macrocycles A1-A50 of the          Present Invention.                                                            Substituents for R.sub.1 -R.sub.6 are provided in TABLE A and for R.sub.7     -R.sub.12 in TABLE B.                                                         TXP                                                                              R.sub.1      R.sub.2                                                                              R.sub.3                                                                              R.sub.4                                                                           R.sub.5                                                                              R.sub.8                              __________________________________________________________________________    A1 CH.sub.2 (CH.sub.2).sub.2 OH                                                               CH.sub.2 CH.sub.3                                                                    CH.sub.2 CH.sub.3                                                                    CH.sub.3                                                                          H      COOH                                 A2 "            "      "      "   "      COOH                                 A3 "            "      "      "   "      CONHCH--(CH.sub.2 OH).sub.2          A4 "            "      "      "   "      "                                    A5 "            "      "      "   "      H                                    A6 "            "      "      "   "      OCH.sub.3                            A7 "            "      "      "   "      "                                    AB "            "      "      "   "      "                                    A9 "            "      "      "   "      "                                    A10                                                                              "            "      "      "   "      "                                    A11                                                                              "            "      "      "   "      "                                    A12                                                                              "            "      "      "   "      "                                    A13                                                                              "            "      "      "   "      CH.sub.3                             A14                                                                              "            "      "      "   "      "                                    A15                                                                              "            "      "      "   "      "                                    A16                                                                              "            "      "      "   "      "                                    A17                                                                              "            "      "      "   CH.sub.3                                                                             H                                    A18                                                                              "            "      "      "   "      "                                    A19                                                                              "            "      "      "   "      "                                    A20                                                                              CH.sub.2 (CH.sub.2).sub.2 OH                                                               CH.sub.2 CH.sub.3                                                                    CH.sub.2 CH.sub.3                                                                    CH.sub.3                                                                          CH.sub.3                                                                             H                                    A21                                                                              "            "      "      "   "      "                                    A22                                                                              "            "      "      "   "      "                                    A23                                                                              "            "      "      "   "      "                                    A24                                                                              "            "      "      "   "      "                                    A25                                                                              "            "      "      "   "      "                                    A26                                                                              "            "      "      "   "      OH                                   A27                                                                              "            "      "      "   "      F                                    A28                                                                              "            "      "      "   CH.sub.2 (CH.sub.2).sub.6 OH                                                         H                                    A29                                                                              "            "      "      "   H      Br                                   A30                                                                              "            "      "      "   "      NO.sub.2                             A31                                                                              "            "      "      "   "      COOH                                 A32                                                                              "            "      "      "   "      CH.sub.3                             A33                                                                              "            "      "      "   C.sub.6 H.sub.5                                                                      H                                    A34                                                                              "            COOH   COOH   "   CH.sub.2 CH.sub.3                                                                    "                                    A35                                                                              "            COOCH.sub.2 CH.sub.3                                                                 COOCH.sub.2 CH.sub.3                                                                 "   CH.sub.3                                                                             "                                    A36                                                                              CH.sub.2 CH.sub.2 CON(CH.sub.2 CH.sub.2 OH).sub.2                                          CH.sub.2 CH.sub.3                                                                    CH.sub.2 CH.sub.3                                                                    "   CH.sub.3                                                                             "                                    A37                                                                              CH.sub.2 CH.sub.2 ON(CH.sub.3)CH.sub.2.                                                    "      "      "   "      "                                       (CHOH).sub.4 CH.sub.2 OH                                                   A38                                                                              CH.sub.2 CH.sub.3                                                                          "      "      "   CH.sub.2 (CH.sub.2).sub.6 OH                                                         "                                    A39                                                                              CH.sub.2 (CH.sub.2).sub.2 OH                                                               CH.sub.2 CH.sub.3                                                                    CH.sub.2 CH.sub.3                                                                    CH.sub.3                                                                          CH.sub.3                                                                             H                                                                      or CH.sub.2 CH.sub.3                        A40                                                                              "            "      "      "   "      "                                    A41                                                                              "            "      "      "   "      "                                    A42                                                                              "            "      "      "   "      "                                    A43                                                                              "            "      "      "   "      "                                    A44                                                                              "            "      "      "   "      "                                    A45                                                                              "            "      "      "   "      "                                    A46                                                                              "            "      "      "   "      "                                    A47                                                                              "            "      "      "   "      "                                    A48                                                                              "            "      "      "   "      "                                    A49                                                                              "            "      "      "   "      "                                    A50                                                                              "            "      "      "   "      "                                    A51                                                                              "            "      "      "   H      "                                    A52                                                                              "            "      "      "   "      "                                    A53                                                                              "            "      "      "   "      "                                    A54                                                                              "            "      "      "   "      "                                    A55                                                                              "            "      "      "   CH.sub.3 or                                                                          "                                                                      CH.sub.2 CH.sub.3                           A56                                                                              "            "      "      "   "      "                                    __________________________________________________________________________

                                      TABLE B                                     __________________________________________________________________________    Representative Substituents for Texaphyrin Macrocycles A1-A50 of the          Present Invention.                                                            Substituents for R.sub.1 -R.sub.6 Are Provided in TABLE A and for R.sub.7     -R.sub.12 in TABLE B.                                                         TXP                                                                              R.sub.7  R.sub.8     R.sub.9  R.sub.10                                                                             R.sub.11                                                                             R.sub.12                       __________________________________________________________________________    A1 O(CH.sub.2).sub.3 OH                                                                   O(CH.sub.2).sub.3 OH                                                                      O(CH.sub.2).sub.3 OH                                                                   H      H      H                              A2 O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                  O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                     COOH     "      "      "                              A3 O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                  O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                     O-saccharide                                                                           "      "      "                              A4 "        "           O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                  "      "      "                              A5 "        O(CH.sub.2).sub.3 CON-linker-oligo                                                        "        "      "      "                              A6 H        OCH.sub.2 CON-linker-oligo                                                                OCH.sub.3                                                                              "      "      "                              A7 "        OCH.sub.2 CO-poly-L-lysine                                                                "        "      "      "                              A8 "        OCH.sub.2 CO-estradiol                                                                    "        "      "      "                              A9 "        O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                     "        "      "      "                              A10                                                                              O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                  "           "        "      "                                     A11                                                                              "        OCH.sub.2 CON-linker-oligo                                                                "        "      "      "                              A12                                                                              "        OCH.sub.2 CO-estradiol                                                                    "        "      "      "                              A13                                                                              "        O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                     O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                  "      "      "                              A14                                                                              "        OCH.sub.2 CO-estradiol                                                                    "        "      "      "                              A15                                                                              O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                  O(CH.sub.2 CH.sub.2 O).sub.120 CH.sub.3                                                   OCH.sub.3                                                                              "      "      "                              A16                                                                              H        saccharide  "        "      "      "                              A17                                                                              O(CH.sub.2).sub.3 OH                                                                   O(CH.sub.2).sub.3 OH                                                                      H        CH.sub.3                                                                             "      "                              A18                                                                              H        O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                     "        "      "      "                              A19                                                                              O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                  "           "        "      "      "                              A20                                                                              H        OCH.sub.2 CON-linker-oligo                                                                H        CH.sub.3                                                                             "      "                              A21                                                                              "        OCH.sub.2 CO-estradiol                                                                    "        "      "      "                              A22                                                                              "        OCH.sub.2 CON(CH.sub.2 CH.sub.2 OH).sub.2                                                 "        "      "      "                              A23                                                                              O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                  O(CH.sub.2 CH.sub.2 O).sub.120 CH.sub.3                                                   "        "      "      "                              A24                                                                              "        OCH.sub.2 CON-linker-oligo                                                                "        "      "      "                              A25                                                                              H        CH.sub.2 CON(CH.sub.3)CH.sub.2.                                                           "        "      "      "                                          (CHOH).sub.4 CH.sub.2 OH                                                                  "        "      "      "                              A26                                                                              O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                  O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                     OH       "      "      "                              A27                                                                              "        "           F        "      "      "                              A28                                                                              "        "           H        CH.sub.2 (CH.sub.2).sub.6 OH                                                         "      "                              A29                                                                              "        "           Br       H      "      "                              A30                                                                              "        "           NO.sub.2 "      "      "                              A31                                                                              "        "           COOH     "      "      "                              A32                                                                              "        "           CH.sub.3 "      "      "                              A33                                                                              "        "           H        C.sub.6 H.sub.5                                                                      "      "                              A34                                                                              "        "           "        CH.sub.2 CH.sub.3                                                                    "      "                              A35                                                                              "        "           "        CH.sub.3                                                                             "      "                              A36                                                                              "        "           "        "      "      "                              A37                                                                              OCH.sub.3                                                                              OCH.sub.3   "        "      "      "                              A38                                                                              H        OCH.sub.2 CO.sub.2 -glucosamine                                                           "        CH.sub.2 (CH.sub.2).sub.6 OH                                                         "      "                              A39                                                                              O(CH.sub.2).sub.3 OH                                                                   O(CH.sub.2).sub.3 OH                                                                      H        CH.sub.3                                                                             CH.sub.3                                                                             CH.sub.3                                                        or     or     or                                                              CH.sub.2 CH.sub.3                                                                    CH.sub.2 CH.sub.3                                                                    CH.sub.2 CH.sub.3              A40                                                                              O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                  O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                     "        "      "      "                              A41                                                                              O(CH.sub.2).sub.3 OH                                                                   O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                     "        "      "      "                              A42                                                                              H        O(CH.sub.2).sub.n CON-linker-oligo,                                                       "        "      "      "                                          n = 1,2,3                                                         A43                                                                              H        O(CH.sub.2).sub.n CO-estradiol,                                                           "        "      "      "                                          n = 1,2,3                                                         A44                                                                              H        saccharide  "        "      "      "                              A45                                                                              O(CH.sub.2).sub.3 OH                                                                   O(CH.sub.2).sub.n CON-linker-oligo,                                                       "        "      "      "                                          n = 1,2,3   "        "      "      "                              A46                                                                              "        O(CH.sub.2).sub.n CO-estradiol,                                                           "        "      "      "                                          n = 1,2,3                                                         A47                                                                              "        saccharide  "        "      "      "                              A48                                                                              O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                  O(CH.sub.2).sub.n CON-linker-oligo,                                                       "        "      "      "                                          n = 1,2,3                                                         A49                                                                              "        O(CH.sub.2).sub.n CO-estradiol,                                                           "        "      "      "                                          n = 1,2,3                                                         A50                                                                              "        saccharide  "        "      "      "                              A51                                                                              "        O(CH.sub.2).sub.n CON-linker-oligo                                                        "        H      "      "                                          n = 1,2,3                                                         A52                                                                              "        O(CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3                                                     "        "      "      "                              A53                                                                              "        "           "        "      CH.sub.2 (CH.sub.2).sub.2                                                            CH.sub.2 (CH.sub.2).sub.2                                                     OH                             A54                                                                              "        O(CH.sub.2).sub.n CON-linker-oligo                                                        "        "      "      "                                          n = 1,2,3                                                         A55                                                                              "        "           "        CH.sub.3 or                                                                          "      "                                                               CH.sub.2 CH.sub.3                            A56                                                                              "        O(CH.sub.2 CH.sub.2 O.sub.).sub.3 CH.sub.3                                                "        "      "      "                              __________________________________________________________________________

A substituent on the R₅, R₁₀, R₁₁ or R₁₂ position of the macrocycle maybe derivatized after condensation of the macrocycle. Substituents mayinclude an alkyl group having up to 5 carbon atoms or a phenyl groupwhich may be further derivatized with a nitro, carboxyl, sulfonic acid,hydroxyl, halide or alkoxy where the alkyl of the alkoxy may behydroxyalkyl and like, as described in U.S. Pat. No. 5,252,720 andapplication Ser. No. 08/135,118.

EXAMPLE 8 Further Derivatives of Texaphyrin

One skilled in the art of organic synthesis in light of the presentdisclosure could extend and refine the basic synthetic chemistryoutlined in this application, in U.S. Pat. No. 5,252,720 and inapplication Ser. No. 08/135,118 so as to produce texaphyrins havingvarious substituents, yet having basic utility to those specificallydetailed in the present examples. For example, polyether-linkedpolyhydroxylated groups, catechol (i.e. benzene diol) derivativesbearing further hydroxyalkyl substituents off the tripyrrane-derivedportion of the macrocycle, saccharide substitutions in which thesaccharide is appended via an acetal-like glycosidic linkage, anoligosaccharide or a polysaccharide may be similarly linked to atexaphyrin. A doubly carboxylated texaphyrin in which the carboxylgroups are linked to the texaphyrin core via aryl ethers orfunctionalized alkyl substituents could be converted to variousesterified products wherein the ester linkages serve to append furtherhydroxyl-containing substituents. Polyhydroxylated texaphyrinderivatives may be synthesized via the use of secondary amide linkages.Saccharide moieties may be appended via amide bonds. Polyhydroxylatedtexaphyrin derivatives containing branched polyhydroxyl (polyol)subunits may be appended to the texaphyrin core via aryl ethers or esterlinkages.

Treatment of carboxylated texaphyrins with thionyl chloride orp-nitrophenol acetate would generate activated acyl species suitable forattachment to monoclonal antibodies or other biomolecules of interest.Standard in situ coupling methods (e.g. 1,1'-carbonyldiimidazole (CDI))could be used to effect the conjugation.

The selectivity of the texaphyrins may be enhanced by covalently linkingoligonucleotides onto the periphery of the macrocycle. Amides, ethersand thioethers are representative of linkages which may be used for thispurpose. Oligonucleotides functionalized with amines at the 5'-end, the3'-end, or internally at sugar or base residues may be modifiedpost-synthetically with an activated carboxylic ester derivative of thetexaphyrin complex. Alternatively, oligonucleotide analogs containingone or more thiophosphate or thiol groups may be selectively alkylatedat the sulfur atom(s) with an alkyl halide derivative of the texaphyrincomplex. The resultant oligodeoxynucleotide-texaphyrin complexconjugates may be designed so as to provide optimal catalyticinteraction between a target nucleic acid and the bound texaphyrin. Theoligonucleotide may be large enough to bind probably at least 15nucleotides of complementary nucleic acid.

A general method for preparing oligonucleotides of various lengths andsequences is described by Caracciolo et al. (1989). Preferredoligonucleotides resistant to in vivo hydrolysis may contain aphosphorothioate substitution at each base (J. Org. Chem., 55:4693-4699,1990). Oligodeoxynucleotides or their phosphorothioate analogues may besynthesized using an Applied Biosystem 380B DNA synthesizer (AppliedBiosystems, Inc., Foster City, Calif.). Specific methods for preparingtexaphyrin-oligonucleotide conjugates are disclosed in PCT publicationWO 94/29316, the disclosure of which is incorporated herein byreference.

Another means of gaining selectivity may be to covalently link thetexaphyrin complex to a sapphyrin (sap) molecule, (U.S. Pat. No.5,159,065; U.S. Pat. No. 5,120,411; U.S. Pat. No. 5,041,078, allincorporated by reference herein.) Since sapphyrins bind DNA, K˜10⁶ M⁻¹,(U.S. Ser. No. 07/964,607, incorporated by reference herein) the linkedtexaphyrin-sapphyrin complex (txph-sap) could effectively increase thetexaphyrin concentration at locations adjacent to the sapphyrin bindingsites. Sapphyrins have a higher fluorescent quantum yield thantexaphyrins, allowing greater fluorescence detection. A laser system maybe employed where the molecules are optimized to the laser wavelength;an excited sapphyrin may transfer its energy to the conjugatedtexaphyrin for detection. The texaphyrin molecule may further bedesigned to pass through cell membranes for selectiveradiosensitization.

New texaphyrin derivatives are characterized fully using normalspectroscopic and analytical means, including, X-ray diffractionmethods.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications in lightthereof will be suggested to persons skilled in the art and are to beincluded within the spirit and purview of this application and scope ofthe appended claims.

What is claimed is:
 1. A method of producing light-induced singletoxygen, the method comprising subjecting a texaphyrin to light in thepresence of oxygen, wherein the texaphyrin has the structure: ##STR14##wherein M is a diamagnetic metal cation;R₁ -R₄, R₇ and R₈ areindependently hydrogen, halide, hydroxyl, alkyl, aryl, haloalkyl, nitro,formyl, acyl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, saccharide,carboxy, carboxyalkyl, carboxyamidealkyl, a site-directing molecule, acatalytic group, or a couple to a site-directing molecule or to acatalytic group; R₆ and R₉ are independently selected from the groups ofR₁ -R₄, R₇ and R₈, with the proviso that the halide is other than iodideand the haloalkyl is other than iodoalkyl; R₅ and R₁₀ -R₁₂ areindependently hydrogen, alkyl, aryl, hydroxyalkyl, oxyalkyl,oxyhydroxyalkyl, carboxyalkyl, carboxyamidealkyl or a couple to asaccharide, to a site-directing molecule or to a catalytic group; atleast one of R₅, R₆, R₉, R₁₀, R₁₁ and R₁₂ is other than hydrogen; and Zis an integer less than or equal to
 5. 2. The method of claim 1 wherethe paramagnetic metal cation is selected from the group consisting ofCd(II), Zn(II), In(III), Y(III), La(III), and Lu(III).
 3. The method ofclaim 1 where the light has a wavelength range from about 730 to about770 nm.
 4. The method of claim 1 where the couple is an amide,disulfide, thioether or ether covalent bond.
 5. The method of claim 1where the catalytic group is selected from the group consisting ofimidazole, guanidine, substituted saccharides, amino acids, derivativesof amino acids, polymers of amino acids, and texaphyrin metal complexes.6. The method of claim 1 where the site-directing molecule has bindingspecificity for localization to a treatment site.
 7. The method of claim1 where the site-directing molecule is selected from the groupconsisting of an oligonucleotide, an antibody, a hormone, a hormonemimic, a peptide having affinity for a biological receptor, and asapphyrin molecule.
 8. The method of claim 1 where at least one of R₅and R₁₀ -R₁₂ is other than hydrogen; andwhen R₅ is other than hydrogen,then R₆ is hydrogen, halide other than iodide, or hydroxyl; and when R₁₀is other than hydrogen, then R₉ is hydrogen, halide other than iodide,or hydroxyl.
 9. The method of claim 1 where at least one of R₆ and R₉ isother than hydrogen; andwhen R₆ is other than hydrogen, then R₅ ishydrogen or methyl; and when R₉ is other than hydrogen, then R₁₀ ishydrogen or methyl.
 10. The method of claim 1 where R₅ and R₁₀ are arylhaving an R₁₃ substituent where R₁₃ is hydrogen, nitro, carboxy,sulfonic acid, hydroxy, oxyalkyl or halide.
 11. The method of claim 1where each of R₁ -R₁₂ is any one of the substituents for R₁ -R₁₂ set outin Tables A and B.
 12. The method of claim 1 where R₁ is CH₂ (CH₂)₂ OH,R₂ and R₃ are CH₂ CH₃, R₄, R₅ and R₁₀ are CH₃, R₆ and R₉ are H, and R₇and R₈ are O(CH₂ CH₂ O)₃ CH₃ or R₇ is H or OCH₃ and R₈ is asite-directing molecule or a couple to a site-directing molecule. 13.The method of claim 12 where R₁₁ and R₁₂ are H or CH₃.
 14. The method ofclaim 1 where the texaphyrin is selected from texaphyrins A1-A56 ofTables A and B.